CN102827941A - Gene chip testing kit for recurrent nasopharyngeal carcinoma - Google Patents
Gene chip testing kit for recurrent nasopharyngeal carcinoma Download PDFInfo
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- CN102827941A CN102827941A CN2012103474104A CN201210347410A CN102827941A CN 102827941 A CN102827941 A CN 102827941A CN 2012103474104 A CN2012103474104 A CN 2012103474104A CN 201210347410 A CN201210347410 A CN 201210347410A CN 102827941 A CN102827941 A CN 102827941A
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Abstract
The invention belongs to the technical field of test of recurrent nasopharyngeal carcinoma and provides a gene chip testing kit for recurrent nasopharyngeal carcinoma. A large quantity of DNA sequences are obtained by collecting deoxyribonucleic acid (DNA) in peripheral blood of a clinical patient, carrying out polymerase chain reaction (PCR) for nucleic acid amplification; the DNA is crossbed with the prepared recurrent-nasopharyngeal-carcinoma-related gene chip kit, and the expression condition of the related gene is obtained by signal scanning, further prediction for recurrent nasopharyngeal carcinoma can be realized. The method provided by the testing kit is simple to operate, convenient and rapid. The kit can be used for effectively testing the change of recurrent gene after nasopharyngeal carcinoma chemoradiotherapy. Medical staff can rapidly and intuitionally predict the possibility of relapse of nasopharyngeal carcinoma after treatment. As the risk of nasopharyngeal carcinoma is predicted, earlier screening and earlier diagnosis are possible, which can improve the survival rate of nasopharyngeal carcinoma patients.
Description
Technical field
The invention belongs to nasopharyngeal carcinoma recurrence detection technique field, relate in particular to a kind of recurrence nasopharyngeal carcinoma gene chip detection kit.
Background technology
Nasopharyngeal carcinoma is China's south common malignancy, is meant the canceration that the mucous membrane of nasopharynx epithelium takes place, and pathology is low differentiated squamous-cell carcinomas mostly; Its grade of malignancy is high; Site of pathological change is hidden, and particularly at pharyngeal recess and nasopharynx top, early symptom is not obvious thereby be difficult to early discovery; The sing misdiagnosis and mistreatment rate is higher, can reach 12.2%.In the nasopharyngeal carcinoma of making a definite diagnosis, its 5 years survival rates are paced up and down about 50%～60% for a long time.Clinical observation for many years and experimental study show that it and inherited genetic factors, Epstein-Barr virus, environmental factors are closely related.The first-selected radiotherapy of present nasopharyngeal carcinoma; After yet even clinical statistics is found the complex therapy of nasopharyngeal carcinoma patient through reasonable radiotherapy and standard, part patient still be difficult to avoid to occur nasopharynx local and (or) tumor recurrence in lymphonodi cervicales drainage district, recurrence rate is still at 8.6-23.7%; Radiotherapeutic effect is relatively poor again; The middle and advanced stage patient of nasopharyngeal carcinoma particularly, survival rate was not ideal enough in 5 years, and therefore many scholars are exploring more effective treat-ment.Although nose can carry out the inspection of each items such as posterior rhinoscope, Epstein-Barr virus serology, iconography (like CT, MRI), Electronic Nose pharyngoscope suspicious patient is carried out the pharynx nasalis biopsy clarifying a diagnosis because of cancerous diagnose clinically, yet lack a kind of simple, effective detection method to curative effect behind the prediction nasopharyngeal carcinoma patient chemicotherapy.
Discover that at present nasopharyngeal carcinoma is a polygene, multistage process, tumour is that several genes is expressed change cumulative result in the cell.Along with the development of molecular biosciences medical technology; Occur utilizing biotechnology to process the kit for screening of utility model in the market; As utilize western technology, the technological external diagnosis reagent cases such as cervical cancer, mammary cancer, the rectum cancer of making of enzyme-linked immunosorbent assay, be used for asymptomatic crowd's precancerous lesion and malignant tumour early screening.
Summary of the invention
The invention provides a kind of recurrence nasopharyngeal carcinoma gene chip detection kit, be intended to solve prior art to prediction nasopharyngeal carcinoma patient chemicotherapy after curative effect but lack a kind of simple, effective problem of detection method.
The object of the present invention is to provide a kind of recurrence nasopharyngeal carcinoma gene chip detection kit, this detection kit is made up of dna probe gene chip, DNA extraction test kit, amplification liquid, hybridization and chromophoric solution to KRT17, S100A8, PI, CLCA2 gene design.
Further, be oligonucleotide fragment to the dna probe of KRT17, S100A8, PI, CLCA2 gene design, and complementary with target DNA to be detected.
Further, said amplification liquid has comprised upstream and downstream primer and the required all ingredients of PCR reaction.
Further, said chromophoric solution comprises prehybridization solution, hybridization buffer, washing lotion 1, washing lotion 2, washing lotion 3, antibody liquid and colour developing liquid.
Further, the dna probe gene chip preparation method to KRT17, S100A8, PI, CLCA2 gene design is:
With the dna probe of KRT17 gene, S100A8 gene, PI gene, CLCA2 gene respectively through specific high speed spotting robot directly on chip.
Further; The high speed spotting robot that is adopted has the printing/jet-printing head of a plurality of printings of a three-dimensional running gear of cover computer control/spray printing pin; A damping base; Give birth to porous plate and a plurality of chip of probe in can putting above, printings/spray printing pin takes out directly printing or spray printing with probe on chip from porous plate.
Recurrence nasopharyngeal carcinoma gene chip detection kit provided by the invention through taking the DNA in the clinical patient peripheral blood, is utilized PCR to carry out nucleic acid amplification and is obtained a large amount of dna sequence dnas; And hybridize with the recurrence nasopharyngeal carcinoma related gene chip agent box for preparing, through signal scanning, obtain the Expression of Related Genes situation; For recurring the prediction that provides of nasopharyngeal carcinoma from now on; This method that this detection kit provides is simple to operate, convenient and swift, can effectively detect the change of recurring gene behind the nasopharyngeal carcinoma chemicotherapy; Let medical personnel predict the possibility of nasopharyngeal carcinoma treatment back recurrence fast, intuitively; Help nasopharyngeal carcinoma forecasting risk and prevent, accomplished examination early, diagnosis early, improved nasopharyngeal carcinoma patient survival rate.
Description of drawings
Fig. 1 is the synoptic diagram of the recurrence nasopharyngeal carcinoma gene chip that provides of the embodiment of the invention.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearer,, the present invention is further specified below in conjunction with accompanying drawing and embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in the qualification invention.
Fig. 1 shows the structure of the recurrence nasopharyngeal carcinoma gene chip that the embodiment of the invention provides.For the ease of explanation, only show the part relevant with the present invention.
The object of the present invention is to provide a kind of recurrence nose because of the oncogene chip inspecting reagent unit, this detection kit is made up of dna probe gene chip, DNA extraction test kit, amplification liquid, hybridization and chromophoric solution to KRT17, S100A8, PI, CLCA2 gene design.
In embodiments of the present invention, be oligonucleotide fragment to the dna probe of KRT17, S100A8, PI, CLCA2 gene design, and complementary with target DNA to be detected.
In embodiments of the present invention, amplification liquid has comprised upstream and downstream primer and the required all ingredients of PCR reaction.
In embodiments of the present invention, chromophoric solution comprises prehybridization solution, hybridization buffer, washing lotion 1, washing lotion 2, washing lotion 3, antibody liquid and colour developing liquid.
In embodiments of the present invention, the dna probe gene chip preparation method to KRT117, S100A8, PI, CLCA2 gene design is:
With the dna probe of KRT17 gene, S100A8 gene, PI gene, CLCA2 gene respectively through specific high speed spotting robot directly on chip.
In embodiments of the present invention; The high speed spotting robot that is adopted has the printing/jet-printing head of a plurality of printings of a three-dimensional running gear of cover computer control/spray printing pin; A damping base; Give birth to porous plate and a plurality of chip of probe in can putting above, printings/spray printing pin takes out directly printing or spray printing with probe on chip from porous plate.
Below in conjunction with accompanying drawing and specific embodiment application principle of the present invention is further described.
To the existing defective of predicting the prognosis of nasopharyngeal carcinoma chemicotherapy clinically, the present invention seeks to propose a kind of method of novel detection nasopharyngeal carcinoma recurrence possibility.At first adopt the dna microarray chip technology; On a large scale, high-throughput ground is to recurrence nasopharyngeal carcinoma patient and just send out thousands of differential genes of nasopharyngeal carcinoma patient and screen, set screening conditions to be: with between group more than 2 times differential expression be condition and P＜0.001 pair recurrence nasopharyngeal carcinoma and just send out a nasopharyngeal carcinoma differential gene and screen.Utilize the accuracy of RT-PCR chip results to verify.For obtaining the gene chip data accuracy; Experiment is carried out the duplicate genes chip detection 3 times to sample; Get the good reproducibility experimental result; Detected result shows that there are a large amount of difference expression genes in a recurrence nasopharyngeal carcinoma and the first nasopharyngeal carcinoma of sending out, wherein screen 104 of the eligible genes relevant with rNPC, 4 maximum differential genes of selection differences multiple from 104 differential genes; Confirm as the relevant gene of recurrence nasopharyngeal carcinoma, carry out clinical detection to these genes involved probe stationary generate a reagent box on chip.This method is simple to operate, and is convenient and swift, can effectively detect the change of recurrence gene behind the nasopharyngeal carcinoma chemicotherapy, lets medical personnel predict possibility of nasopharyngeal carcinoma treatment back recurrence fast, intuitively, help nasopharyngeal carcinoma forecasting risk and preventing.Accomplishing examination early, diagnosis early, is most important to improving nasopharyngeal carcinoma patient survival rate.
(1) recurrence nasopharyngeal carcinoma differential gene screening
1, sample source and experiment are divided into groups
Is research object just to send out nasopharyngeal carcinoma with sending out nasopharyngeal carcinoma; Recurrence nasopharyngeal carcinoma patient for radiotherapy first after 2～6 years generation local recurrence, pathology is made a definite diagnosis and is PDSCC, does not find metastasis on inspection; Those selected is at electronics nasopharyngeal fiberscope direct vision biopsy, and biopsy divides 2 parts, and 1 part is sent pathologic finding, and is frozen subsequent use in 1 part of nitrogen of tapping at once;
2,, judge the high-quality total RNA that is that is extracted with spectrophotometer measurement total rna concentration and purity; 3, probe mark
Utilize reverse transcriptional PCR PCR, after the two-wheeled rt became c DNA, purifying cDNA and sectionization single stranded DNA sheet added reagent in the DNA of segmentization, and mixing is got 2 μ l sample detection labeling effciencies.Hybridize then, wash, in the 1.5ml centrifuge tube, prepare hybridization solution, mixing, centrifugal, in Affymetrix Fluidics Station450 wash plant, wash every routine sample triplicate then;
4, scanning and analysis
The setting screening conditions are: with the group between more than 2 times differential expression be condition and P＜0.001; Respectively pNPC and rNPC difference expression gene are screened, confirm that from 104 differential genes KRT17, S100A8, PI, CLCA2 change may cause the nasopharyngeal carcinoma recurrence;
(2) preparation of recurrence nasopharyngeal carcinoma gene chip detection kit
1, test kit is formed
Gene chip kit is respectively by the dna probe (oligonucleotide fragment to KRT17, S100A8, PI, CLCA2 gene design; Complementary with target DNA to be detected) gene chip, DNA extraction test kit, amplification liquid (comprised required all ingredients of upstream and downstream primer and PCR reaction such as dNTP, Mgcl2, H2O, Buffer etc., Taq enzyme pipe in addition provides), hybridization and chromophoric solution components such as (comprising prehybridization solution, hybridization buffer, washing lotion 1, washing lotion 2, washing lotion 3, antibody liquid and colour developing liquid) form.
2, make the chip of recurrence nasopharyngeal carcinoma gene DNA probe
Probe generally is to choose a certain gene fragment in the gene library; Be connected with plasmid or phage vector clone after; Obtain the recombinant plasmid of this gene; In being transformed into suitable bacterial body, the plasmid of extraction is received the specific gene fragment through the restriction enzyme switchback, then becomes genome DNA probe through mark.
With the KRT17 gene (the NCBI accession number: NM_000422.2), the S100A8 gene (the NCBI accession number: NM_002964.3), the PI gene (the NCBI accession number: BC010952), the CLCA2 gene-based because of (the NCBI accession number: dna probe NM_006536) respectively through specific high speed spotting robot directly on chip.There are the printing/jet-printing head of a plurality of printings of a three-dimensional running gear of cover computer control/spray printing pin, a damping base, porous plate and a plurality of chip of living probe in can putting above in the robot that adopts.Printing/spray printing pin takes out directly printing or spray printing with probe on chip from porous plate.
(3) the recurrence nasopharyngeal carcinoma detects step
1, the extraction of recurrence nasopharyngeal carcinoma patient DNA
Extract the genomic dna in the recurrence nasopharyngeal carcinoma peripheral blood; Add a certain amount of Virahol or ethanol; Genomic macromole DNA promptly precipitates the fibrous floc sedimentation of formation and floats wherein; Available glass rod takes out it, and small molecule DNA then only forms the particulate state deposition and invests on the wall and the bottom, thereby reaches the purpose of extraction.
2, DNA cloning
With amplification add the Taq enzyme in the liquid, target DNA that extracting is good enlarges target dna fragment rapidly through the PCR reaction, is used for hybridization and coupling reaction.
3, hybridization and colour developing
The gene chip hybridization reaction is to carry out specific hybrid through the dna probe to KRT17, S100A8, PI, CLCA2 gene design on target DNA and the gene chip, again through the coupling reaction colour developing.
4, detect
Color spot on the gene chip just can be known mononucleotide in the sample DNA sequence through the signal amplification, just can confirm whether exist, just explain that there is the recurrence possibility in the patient if exist with recurrence nasopharyngeal carcinoma related gene KRT17, S100A8, PI, CLCA2.
The recurrence nasopharyngeal carcinoma gene chip detection kit that the embodiment of the invention provides through taking the DNA in the clinical patient peripheral blood, is utilized PCR to carry out nucleic acid amplification and is obtained a large amount of dna sequence dnas; And hybridize with the recurrence nasopharyngeal carcinoma related gene chip agent box for preparing, through signal scanning, obtain the Expression of Related Genes situation; For recurring the prediction that provides of nasopharyngeal carcinoma from now on; This method that this detection kit provides is simple to operate, convenient and swift, can effectively detect the change of recurring gene behind the nasopharyngeal carcinoma chemicotherapy; Let medical personnel predict the possibility of nasopharyngeal carcinoma treatment back recurrence fast, intuitively; Help nasopharyngeal carcinoma forecasting risk and prevent, accomplished examination early, diagnosis early, improved nasopharyngeal carcinoma patient survival rate.
More than be merely preferred embodiment of the present invention,, all any modifications of within spirit of the present invention and principle, being done, be equal to and replace and improvement etc., all should be included within protection scope of the present invention not in order to restriction the present invention.
Claims (6)
1. a recurrence nasopharyngeal carcinoma gene chip detection kit is characterized in that, this detection kit is made up of dna probe gene chip, DNA extraction test kit, amplification liquid, hybridization and chromophoric solution to KRT17, S100A8, PI, CLCA2 gene design.
2. detection kit as claimed in claim 1 is characterized in that, is oligonucleotide fragment to the dna probe of KRT17, S100A8, PI, CLCA2 gene design, and complementary with target DNA to be detected.
3. detection kit as claimed in claim 1 is characterized in that, said amplification liquid has comprised upstream and downstream primer and the required all ingredients of PCR reaction.
4. detection kit as claimed in claim 1 is characterized in that, said chromophoric solution comprises prehybridization solution, hybridization buffer, washing lotion 1, washing lotion 2, washing lotion 3, antibody liquid and colour developing liquid.
5. detection kit as claimed in claim 1 is characterized in that, to the dna probe gene chip preparation method of KRT17, S100A8, PI, CLCA2 gene design is:
With the dna probe of KRT17 gene, S100A8 gene, PI gene, CLCA2 gene respectively through specific high speed spotting robot directly on chip.
6. detection kit as claimed in claim 5; It is characterized in that; The high speed spotting robot that is adopted has the printing/jet-printing head of a plurality of printings of a three-dimensional running gear of cover computer control/spray printing pin; A damping base is given birth to porous plate and a plurality of chip of probe in can putting above, printings/spray printing pin takes out directly printing or spray printing with probe on chip from porous plate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108198621A (en) * | 2018-01-18 | 2018-06-22 | 中山大学 | A kind of database data synthesis dicision of diagnosis and treatment method based on neural network |
| CN114032339A (en) * | 2021-12-22 | 2022-02-11 | 湖南工程学院 | Hyperbranched hybridization chain reaction signal amplification system, kit and detection method for detecting nasopharyngeal carcinoma |
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| CN101333561A (en) * | 2007-06-29 | 2008-12-31 | 上海裕隆生物科技有限公司 | Detecting kit for cardiovascular and cerebrovascular diseases predisposing gene chip |
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- 2012-09-06 CN CN2012103474104A patent/CN102827941A/en active Pending
Patent Citations (1)
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| CN101333561A (en) * | 2007-06-29 | 2008-12-31 | 上海裕隆生物科技有限公司 | Detecting kit for cardiovascular and cerebrovascular diseases predisposing gene chip |
Non-Patent Citations (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108198621A (en) * | 2018-01-18 | 2018-06-22 | 中山大学 | A kind of database data synthesis dicision of diagnosis and treatment method based on neural network |
| CN108198621B (en) * | 2018-01-18 | 2022-03-08 | 中山大学 | Database data comprehensive diagnosis and treatment decision method based on neural network |
| CN114032339A (en) * | 2021-12-22 | 2022-02-11 | 湖南工程学院 | Hyperbranched hybridization chain reaction signal amplification system, kit and detection method for detecting nasopharyngeal carcinoma |
| CN114032339B (en) * | 2021-12-22 | 2023-09-01 | 湖南工程学院 | Hyperbranched hybridization chain reaction signal amplification system for detecting nasopharyngeal carcinoma, kit and detection method |
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Application publication date: 20121219 |